Method for conserving biological prostheses, conserved biological prostheses and conserving solutions

ABSTRACT

The invention concerns a method of conserving biological prostheses, wherein the method includes the following steps: (a) treating biological prostheses with a solution which contains a mixture of epoxide compounds which are at least in part of different lengths; (b) treating the biological prosthesis treated in accordance with step (a) with an antithrombotic-bearing solution; and (c) possibly storing the prosthesis treated in accordance with step (b) in a sterilising solution. The invention further concerns a biological prosthesis produced in accordance with the method of the invention and a conserving agent.

[0001] The invention concerns a method of conserving biologicalprostheses and biological prostheses produced in accordance with thatmethod. The invention further concerns a conserving solution forconserving biological prostheses.

[0002] Biological prostheses can be produced from body components ofhuman beings and animals. For example heart valves, arteries etc ofcattle or pigs are used as implants in human beings.

[0003] It will be appreciated that the biological prostheses must bechemically treated prior to implantation in the organism of the humanbeing. The treatment method must ensure:

[0004] 1) the absence of immunogenity (that applies equally toheterologous and allogenous tissue);

[0005] 2) sterility of the implant;

[0006] 3) high strength, elasticity and deformation properties of thebiological material; and

[0007] 4) high biocompatibility whose main parameter, in relation to thebiological prostheses, is the absence of calcification and thromboses inthe event of long-term use in the recipient organism.

[0008] For more than 30 years now glutaraldehyde has represented themain conserving agent for cardiac-circulatory bioprostheses (biologicalprostheses). Glutaraldehyde reacts with the amino groups of lysine andhydroxylysine. As a result of those reactions, chemical bonds areformed, which are represented in particular by Schiff's bases orpyridine bases. Glutaraldehyde ensures reliable sterility and thesuppression of antigenic properties of the biological material.

[0009] Glutaraldehyde however makes the tissue stiff and hydrophobic andthe surface assumes a rough disordered relief. In addition the chemicalcompounds of glutaraldehyde and collagen have in their structure ligandsfor complex formation with calcium cations. Those complexes do in factsubsequently become centers of hydroxyapatite crystallisation. Inoverall terms the result of this is that the glutaraldehyde has anegative influence on the thrombosis-resistant properties of the tissueand gives rise to calcification of the biological materials.

[0010] In order to eliminate the negative influence of glutaldehyde onthe biological material, methods have been developed for the additionalchemical modification of biological prostheses.

[0011] For increasing thrombosis resistance use was made for example ofheparin (U.S. Pat. No. 3,988,782) while aminodiphosphonates were usedfor the prophylaxis of calcification (U.S. Pat. No. 4,553,974). Howeverthose methods do not have the expected effect as the negative influenceof the glutaraldehyde as a main conserving agent on the tissue isexcessively great.

[0012] A greater effect can be achieved by replacing the main conservingagent by a cross-linking agent, the structural formula of which does notcontain any aldehyde group.

[0013] Methods are known for conserving biological prostheses usingindividual polymer (U.S. Pat. No. 4,806,595 and U.S. Pat. No. 5,080,670)and non-polymer (U.S. Pat. No. 5,880,242 and RU 2 008 767) epoxidecompounds.

[0014] Epoxide compounds are effective cross-linking agents, guaranteestrength and elasticity of the biological material, inhibitcalcification of the biological prostheses and have pronouncedantigen-depressive properties.

[0015] To improve the thrombosis resistance of the tissue, it ispossible to implement an additional modification with heparin (U.S. Pat.No. 4,806,595 and RU 2 008 767). The disadvantages of those methods areas follows:

[0016] 1) A masking effect which is characteristic of non-polymer orpolymer di-epoxides when one of the epoxy groups reacts with collagenbut the other remains without a bond, that is to say it does not react.On the one hand those unbound, that is to say free epoxy groups exhibita cytotoxic effect, on the other hand they result in insufficientdensity in terms of transverse cross-linking, which has a negativeinfluence on the strength properties of the tissue.

[0017] 2) The use of individual polymer epoxides, in the structuralformula of which there are contained more than two epoxide groups,reduces the masking effect but it detrimentally affects transversecross-linking and increases the content of free epoxide groups in thetissue. Without an additional modification which is targeted at closingor converting those groups, on the one hand the cytotoxic effect isincreased. On the other hand the number of such epoxide groups is toolow for saturation of the biological material with substances whichimpart additional properties to the biological prostheses (heparin,anti-bacterial agents and so forth).

[0018] The method known from U.S. Pat. No. 4,806,595 for heparintreatment of epoxy-conserved biological prostheses includes the use ofprotamine which is firmly bound to the collagen by an epoxide bond. Theprocedure involves in turn fixing on the protamine heparin which,lifting off the surface, exerts an anti-coagulant function.

[0019] The effectiveness of such a method is however limited in respectof time and requires the use of an intermediate reagent, namelyprotamine. The possibility of bonding protamine to the collagen—if thisis involves collagen from the bonding tissue of the biologicalprosthesis—is in turn limited by the number of free reactive groups ofthe conserving agent as the main proportion of those groups is requiredfor the cross-linking effect.

[0020] U.S. Pat. No. 5,165,919 discloses a method of covalent fixing ofheparin on medical implants by the interaction of amino and epoxygroups. That method however was developed for polymer materials andcannot be used for biological prostheses.

[0021] The object of the invention is to provide a method of conservingbiological prostheses which are resistant to calcification andthrombosis formation and have an enhanced level of strength andelasticity. Another object of the invention is to provide improvedconserved biological prostheses.

[0022] The object of the invention is attained by a method having thefeatures of claim 1. Preferred developments are recited in appendantclaims 2 to 19.

[0023] The object of the invention is further attained by a biologicalprosthesis as set forth in claim 20. A preferred embodiment of thebiological prosthesis is recited in appendant claim 21.

[0024] The object is further attained by the provision of a conservingagent for biological prostheses as set forth in claim 22. Preferreddevelopments are recited in appendant claims 23 to 27.

[0025] In accordance with the invention preferably mixtures of polymerand non-polymer epoxide compounds are used for conserving biologicalprostheses, wherein 2 and more epoxide groups are present in thestructural formula of the epoxide compounds.

[0026] Preferably a solution with a mixture of at least three differentepoxide compounds (components) is used.

[0027] In a further preferred feature the antithrombotic is selectedfrom the group which consists of heparin, low-molecular heparin,heparinoids, hirudin and mixtures thereof.

[0028] It is essential in accordance with the invention that theantithrombotic has antithrombotic or anti-coagulating properties. Inthat respect it is possible in accordance with the invention to use allsubstances which have a heparin-like action.

[0029] Thus it is also possible to use chemically, mechanically and/orenzymatically modified heparin, truncated heparin, recombinant heparinor mixtures thereof insofar as the correspondingly modified heparin hasantithrombotic properties.

[0030] Preferably heparin is used in accordance with the invention.

[0031] It was surprisingly found that:

[0032] 1) the strength of the biological prosthesis is increased bytransverse connections as the accessibility of reactive groups ofcollagen in the biological prosthesis for an interaction with variousconserving agents which have various molecule lengths is guaranteed.That enhances the strength of the biological material and ensures abetter antigen depression effect;

[0033] 2) the use of compounds with more than 2 epoxide groups in themixture of epoxide compounds permits the production of biologicalmaterial with a predetermined number of free epoxy groups;

[0034] 3) subsequently heparin can be immobilised on those groups toimpart thrombosis resistance. Immobilisation of heparin in the complexwith acetylsalicylic acid strengthens the thrombosis-resistance effect.

[0035] After conservation and modification with heparin the biologicalprostheses produced in that way can be stored in a solution of anycompound which guarantees sterility. Preferably epoxide compounds areused in order to avoid possible additional chemical reactions.

[0036] The subject-matter of the present invention is thus on the onehand a method of conserving biological material which can be used inparticular for producing heart and blood vessel valve prostheses. On theother hand the subject-matter of the invention is the provision of thebiological prostheses which are conserved in accordance with the methodof the invention.

[0037] In accordance with the invention, preferably to provide theconserving effect instead of individual epoxide conserving agents,mixtures of polymer and non-polymer epoxide compounds with a differingnumber of epoxide groups (2 and more) are used. The composition of themixture can be varied in dependence on the nature of the biologicalmaterial and the aims of a subsequent chemical modification.

[0038] Preferably the epoxide group in the epoxide compounds used is aconstituent of a glycidol residue.

[0039] In order at the same time to improve the thrombosis-resistanceproperties and to close, that is to say transform, the free epoxidegroups which have not bound to the collagen in the conservationprocedure subsequent treatment of the biological material with heparinand acetylsalicylic acid is implemented.

[0040] Preferably a mixture of polymer and non-polymer epoxide compoundsis used for conserving heart-circulatory bioprostheses(bioprosthesis—biological prosthesis).

[0041] In accordance with the invention the term a polymer epoxidecompound is used to identify an epoxide compound which is composed of atleast two repetitive, directly interconnected units to which epoxygroup-bearing residues such as for example glycidol or2,3-epoxypropan-1-ol are joined.

[0042] Preferably the polymer epoxide compounds have a degree ofpolymerisation of at least two, more preferably between three and 25,still more preferably between three and 15, in particular between fourand nine. In this respect the degree of polymerisation relates to thepolymer proportion of the epoxide compound, bearing the epoxy groups.

[0043] Instead of a polymer epoxide compound it is also possible to usean epoxide compound with between two and three epoxide groups, whereinarranged between at least two epoxide groups is a straight-chain orbranched hydrocarbon chain with at least four carbon atoms, for examplea tetramethylene group. Preferably arranged between the at least twoepoxide groups is a straight-chain or branched hydrocarbon chain with atleast six, preferably six, carbon atoms, such as for example ahexamethylene group.

[0044] In accordance with the invention the term non-polar epoxidecompound is used to identify an epoxide compound which does not have anyrepetitive, directly interconnected units to which epoxy group-bearingresidues such as for example glycidol or 2,3-epoxypropan-1-ol arejoined.

[0045] It may be advantageous to vary the composition of the mixture independence on the association in respect of nature and tissue of thebiological material. That is related to the spatial configuration andthe composition of the collagens of the various tissues and thedifferent accessibility of the reactive amino acid groups of thecollagen for the conserving agents which have different structuralformulae.

[0046] In addition the composition of the mixture depends on the purposeof the additional chemical modification; saturation of the biologicalmaterial with a predetermined number of epoxide groups which arerequired for covalent immobilisation of biologically active substancesis possible. Thus for example upon immobilisation of heparin a largenumber of epoxide groups is required.

[0047] For that purpose the mixture is preferably adjusted as follows:

[0048] 40-80% by weight, more preferably 50-70% by weight, of at leastone non-polymer epoxide compound with two epoxide groups;

[0049] 5-20% by weight, more preferably 10-15% by weight, of at leastone polymer epoxide compound with between two and three epoxide groupsand/or at least one epoxide compound with between two and three epoxidegroups, wherein arranged between at least two epoxide groups is astraight-chain or branched hydrocarbon chain with at least four carbonatoms; and

[0050] 15-45% by weight, more preferably 20-35% by weight, of at leastone epoxide compound with at least three epoxide groups;

[0051] wherein the total amount is 100% by weight. The foregoingpercentage details of the respective epoxide compound in percent byweight relate in each case to the total amount of epoxide compoundsused.

[0052] In a modification of high-molecular polymer substances incontrast the number of reactive groups must be low as supersaturation ofthe tissue of the biological prosthesis with high-molecular polymersresults in an impairment of the properties in respect of elasticity anddeformation. Accordingly use of the epoxide mixtures of differingcomposition permits the amount of substance which is immobilised on thebiological material to be controlled or influenced.

[0053] For that purpose the mixture is preferably adjusted as follows:

[0054] 70-90% by weight, more preferably 75-85% by weight, of at leastone non-polymer epoxide compound with two epoxide groups;

[0055] 5-20% by weight, more preferably 10-15% by weight, of at leastone polymer epoxide compound with between two and three epoxide groupsand/or at least one epoxide compound with between two and three epoxidegroups, wherein arranged between at least two epoxide groups is astraight-chain or branched hydrocarbon chain with at least four carbonatoms; and

[0056] up to 5% by weight, more preferably 1-2% by weight, of at leastone epoxide compound with at least three epoxide groups;

[0057] wherein the total amount is 100% by weight. The foregoingpercentage details of the respective epoxide compound in percent byweight relate in each case to the total amount of epoxide compoundsused.

[0058] In the case of the above-specified mixtures a non-polymer,preferably low-molecular compound, with two reaction groups, must bepresent in the mixture. For example it is possible to use alkane dioldiglycidylether such as for example butane-1,4-diol diglycidylether,polyalcohol diglycidylether such as for example glycerinediglycidylether, alkylene glycol diglycidylether such as ethylene glycoldiglycidylether or mixtures thereof. Preferably ethylene glycoldiglycidylether is used.

[0059] Those compounds ensure intermolecular transverse cross-linking ofthe collagen and intramolecular cross-linking of proteoglycans andproteins of the cell elements which represent the most active antigenicdeterminants.

[0060] The concentration of non-polymer epoxide compound with twoepoxide groups (di-epoxide compound) in the mixture is preferably 1-95%by weight, more preferably 20-90% by weight. Very good results wereobtained with a concentration of 40-80% by weight.

[0061] The second component in the mixture is a polymer epoxide compoundwhich contains between two and three epoxide groups in the structuralformula and/or an epoxide compound with between two and three epoxidegroups, wherein arranged between at least two epoxide groups is astraight-chain or branched hydrocarbon chain with at least four carbonatoms.

[0062] For example it is possible to use polyalkylene glycoldiglycidylethers such as for example polyethylene glycoldiglycidylether, polytetramethylene glycol diglycidylether,polypropylene glycol diglycidylether or mixtures thereof.

[0063] Instead of or in addition to the above-noted polymer epoxidecompound it is also possible to use alkane diol diglycidylethers such asfor example hexane-1,6-diol diglycidylether and/or higher dicarboxylicacid diglycidylesters.

[0064] In accordance with the invention the term higher dicarboxylicacid diglycidylester is used to identify glycidol esterified with higherdicarboxylic acids (2,3-epoxypropan-1-ol), that is to saydiglycidylesters of higher dicarboxylic acids. Identified as higherdicarboxylic acids are those which have more than 12 carbon atoms.

[0065] By way of example higher dicarboxylic acid diglycidylesters whichcan be used are Denacol EX-1111 (mixture of two acids with a molecularweight of 398 g/mol and 454 g/mol) or Denacol EX-1112 (mixture of twoacids with the same molecular weight of 450 g/mol but of differingstructure) from Nagase Company Ltd, Japan.

[0066] Preferably polyethylene glycol diglycidylether is used.Preferably the polyethylene glycol diglycidylether has a degree ofpolymerisation of 3-12, more preferably 4-9.

[0067] That component supplements cross-linking of the collagen byinter-fibrillar bonds. The concentration in the mixture is preferably1-95%, more preferably 2-40% by weight, still more preferably 5-20% byweight.

[0068] The third component is an epoxide compound with a number ofepoxide groups of at least three, for example four, five or more.

[0069] Preferably in that respect polyalcohol polyglycidylethers areused such as for example sorbitol polyglycidylether, glycerinepolyglycidylether and the like, polysaccharide polyglycidylether ormixtures thereof. Preferably pentaerythrol polyglycidylether is used.

[0070] That third component saturates the tissue with an optimum numberof free epoxide groups, to which heparin can be immobilised without theuse of additional reagents. The concentration of the component in themixture is preferably 1-95% by weight, more preferably 10-50% by weight.Very good results were obtained with a concentration of 15-35% byweight.

[0071] The foregoing percentage details of the respective epoxidecompound in percent by weight relate respectively to the total amount ofepoxide compounds used.

[0072] The method according to the invention permits an increase in thedensity of cross-linking of the collagen, which has an advantageouseffect on the strength properties of the biological prosthesis. The useof mixtures of polymer and non-polymer epoxides imparts to thebiological material a higher level of resistance to calcification.

[0073] Saturation of the tissue with free epoxide groups makes itpossible to implement covalent immobilisation of heparin on thebiological prosthesis when pre-treated in that way.

[0074] In relation to the functional groups of the heparin, the epoxygroup represents one of the most reactive groups in the polymers,including also in biological materials. Binding of the heparin by way ofthe amino group does not have a disadvantageous effect on theanti-coagulant properties thereof.

[0075] Reaction with the epoxy group makes it possible to implementimmobilisation of heparin under ‘gentle’ conditions which excludeunwanted physical-chemical changes in the biological material. Reactionof heparin with the epoxy groups can take place in a wide pH-range ofthe medium (pH 2-11).

[0076] In terms of modification of the biological tissue, preferably apH-value of 5-8 is used, for example employing phosphate buffers,phosphate-citrate buffers and/or acetylsalicylic acid.

[0077] Such an operating procedure makes it possible on the one hand toachieve an improvement in the thrombosis-resistant properties and on theother hand to neutralise the free epoxy groups which are always presentin the biological material as a result of the masking effect.

[0078] The introduction of a third component, that is to say a compoundwith a number of epoxy groups of at least 3, into the epoxide conservingagent makes it possible to increase the amount of immobilised heparin.The covalently bound, that is to say immobilised heparin does not passinto the bloodstream but has an antithrombotic effect due to binding andsorption to the protein layers and smoothing of the surface of thebiological prosthesis.

[0079] The use of acetylsalicylic acid for setting an acid pH-value inthe immobilisation of heparin surprisingly further increases thethrombosis resistance of the surface.

[0080] To produce a conserved biological prosthesis according to theinvention for heart-circulation surgery, such as for example biologicalheart valve prostheses, it is possible to use for example aortacomplexes of a pig or pericardia of cattle. In the case ofarteriosclerosis for example damaged arteries can be replaced by theinternal thoracic artery or the head artery of cattle.

[0081] Allogenous or heterologous mitral valves or tricuspidal valvescan be used for the orthotopic replacement of heart valves. Forintracardial plastic surgery and angioplastic surgery it is possible touse pericardia from cattle, the allogenous hard meninges orbody-specific pericardia. Valves, membrane-like tissues and vessels, forexample arteries, veins, artery segments, vein segments and so forth canbe taken from any biological genuses of mammals, for example cattle,pigs, sheep, human beings and so forth, insofar as they are suitable inrespect of their anatomical features for replacement of one element oranother of the heart-circulation system.

[0082] Examples 1 through 3 according to the invention which are set outhereinafter serve only to further illustrate the invention and in no waylimit the scope of protection thereof.

[0083] In the examples according to the invention a mixture of ethyleneglycol diglycidylether (DEE), pentaerythrol polyglycidylether andpolyethylene glycol diglycidylether were used in the respectivelyspecified quantitative ratios for conservation of the respectivebiological prosthesis used.

[0084] In that respect the examples were all implemented at ambienttemperature, that is to say at between about 20° C. and about 25° C. Itis also possible to use higher temperatures, but not more than 37° C. Inall incubation steps, the solutions were not agitated. It will beappreciated however that the solutions can also be agitated.

EXAMPLE 1 ACCORDING TO THE INVENTION

[0085] Aortic valve vela of a pig (15 g of moist tissue) were rinsedwith 0.9% by weight NaCl solution and put into 200 ml of a conservingsolution according to the invention, which is produced from 50 mMphosphate buffer pH 7.4 and contains 6 g of ethylene glycoldiglycidylether, 1 g of polyethylene glycol diglycidylether (n=5) and 3g of pentaerythrol polyglycidylether (number of glycidylether units permolecule: 4).

[0086] After 48 hours the conserving solution was replaced by anidentical but freshly produced solution. After 12 days the valve velawere rinsed with sterile 0.9% by weight NaCl solution and incubated in aheparin solution (100 IU/ml, IU: International Unit) pH 5.0, in whichcase the pH-value was adjusted by the addition of aqueousacetylsalicylic acid solution, for a period of 3 hours at a temperatureof 20° C.

[0087] Thereafter rinsing was effected five times with an excess of 0.9%by weight NaCl solution and the treated valve vela were put into 5% byweight ethylene glycol diglycidylether solution where they were storeduntil further use.

[0088] Production of the heparin solution

[0089] The ratio of heparin: acetylsalicylic acid in the heparinsolution, in relation to weight, is about 1.3-16:1. The precise ratio ofheparin: acetylsalicylic acid depends on the respective activity (inIU/weight) of the heparin used.

[0090] By way of example a heparin solution which is suitable for theinvention can be produced by the addition of about 7-10 mg/ml ofacetylsalicylic acid to a heparin solution with about 75-100 IU/ml. Inthat case acetylsalicylic acid is added to the heparin solution untilthe pH-value of the solution is between about 5 and 6. The concentrationof the heparin solution is at least 75 IU/ml, preferably 100 IU/ml. Itwill be appreciated that it is also possible to use solutions withhigher heparin concentrations.

COMPARATIVE EXAMPLES 1

[0091] Comparative samples involved using aortic valve vela of a pig,which were treated with 0.625% by weight glutaraldehyde (GA) in 50 mMphosphate buffer pH 7.4 or with 5% by weight ethylene glycoldiglycidylether (DEE) and 100 IU/ml heparin in 50 mM phosphate buffer pH7.4 (DEE+heparin) (see RU 2 008 767).

[0092] a) Treatment of Samples with Glutaraldehyde

[0093] The comparative samples were incubated for 28 days in 0.625% byweight glutaraldehyde (GA), and 50 mM phosphate buffer pH 7.4 at ambienttemperature (between 20° C. and 25° C.). The GA-solution was changedfour times, that is to say after the 1st, 3rd, 7th and 21st days. Priorto further use (analysis or implantation) the conserving solution wasremoved. Subsequently the comparative sample was washed at ambienttemperature for one hour without agitation in 0.9% common salt solution.The common salt solution was changed in that procedure after every 20minutes (a total of three washing steps).

[0094] b) Treatment of Samples with DEE and Heparin

[0095] The comparative sample was incubated for 21 days in 5% by weightethylene glycol diglycidylether (DEE) and 50 mM phosphate buffer pH 7.4at ambient temperature (20° C.-25° C.). The DEE solution was changedafter three days without a washing step. The conserving solution wasthen removed. The comparative sample was then washed at ambienttemperature for one hour without agitation in 0.9% common salt solution.In that procedure the common salt solution was changed after every 20minutes (a total of three washing steps). The heparin treatment waseffected with 100 IU/ml at 37° C. for 8-16 hours. The unbound heparinwas removed by washing with 0.9% by weight common salt solution for onehour at ambient temperature (20-25° C.). During the washing operationthe common salt solution was not agitated and not changed. Thecomparative sample was stored in 2-5% by weight DEE solution. Theconserving solution was removed prior to further use (analysis orimplantation). The comparative example was then washed at ambienttemperature for one hour without agitation in 0.9% common salt solution.In that procedure the common salt solution was changed after every 20minutes (a total of three washing steps).

[0096] Comparison of Examples According to the Invention and ComparativeExamples

[0097] The improvement in the properties of biological prosthesesconserved using the method according to the invention (Examples 1through 3 according to the invention) is clearly shown by comparison ofbiological prostheses conserved with conventional conservation methods(comparative Examples 1 through 3). In that respect the density oftransverse cross-linking of amino acids in the respective biologicalprostheses used, the properties in respect of elasticity anddeformation, the degree of calcification and the amount of immobilisedheparin were determined and compared to each other.

[0098] The above-indicated parameters were determined in that respect inthe following fashion.

[0099] Determining Transverse Cross-Linking

[0100] The density of transverse cross-linking was assessed inaccordance with the reduction in the number of free amino acid residuesin the biological material, in which respect they were determined by anamino acid analysis method.

[0101] For the amino acid analysis procedure, five samples were taken ineach case and washed with distilled water which was renewed twice in onehour. The samples were then lyophilised for three hours (temperature ofthe sample: between −55° C. and +60° C.). Between 1.5 and 2 mg of drytissue was put into between 0.15 ml and 2 ml of 6 N HCl and incubated insealed vacuum flasks for 24 hours at 110° C. Thereafter the acid wasevaporated and the residue was diluted in 2.5 ml of lithium citratebuffer and centrifuged. The supernatant substance was subjected to anamino acid analysis operation in an amino acid analyser (CL 5001BIOTRONIC, Germany) with computer-aided data evaluation (CR-3A, SHIMADZUIntegrator, Japan).

[0102] Determining the Properties in Respect of Elasticity andDeformation

[0103] The samples were cut out in dumbbell shape (dumbbell test bodies)using an especially shaped blade. That blade is of a standard shape andsize and has sharpened edges. Using that blade, samples of astandardised size are cut out of the biological material. Ten sampleswere investigated in each case.

[0104] The properties in respect of elasticity and deformation weredetermined on the tensile strength testing machine ‘Instron-1122’(manufacturer: INSTRON, England).

[0105] All the materials investigated were loaded to investigate thebreaking/tearing strength at a constant speed (50 mm/min).

[0106] The average thickness h of the samples (mm) was determined usinga micrometer eyepiece.

[0107] The data were calculated as follows:

[0108] a) Maximum tensile strength (tensile stress) σ (kg/cm²)

[0109] σ=P_(max)/S, wherein P_(max) is the breaking load (kg) and S isthe cross-sectional area of the sample (cm²),

[0110] S=h×b₀, wherein h (cm) is the thickness of the sample and b₀ (cm)is the width of the sample.

[0111] In the present case the width of the sample was set to 0.25 cmusing the above-mentioned blade.

[0112] b) Maximum stretch ε_(max) (%)

[0113] ε_(max)=(ΔI_(max)/I₀)×100,

[0114] ΔI_(max)=I_(max)−I₀, wherein ‘I_(max)’ is the final length and‘I₀’ is the initial length of the sample.

[0115] In the present case the samples were of an initial length of 11mm.

[0116] Determining Calcification

[0117] Resistance to calcification was investigated by subcutaneousimplantation of conserved biological prostheses under the skin of 3-weekmale rats (diameter of the samples: 7 mm).

[0118] Three samples, in each case one of the biological samplesconserved using GA, DEE+heparin and the conserving solution according tothe invention, were each implanted into a respective one of male Vistarrats (n=33), weight 48.64±3.5 g, under ether anesthesia. The implantswere removed from 11 animals in each case after a respective period of30, 60 and 90 days.

[0119] After removal of the samples they were cleaned of the surroundingtissue, washed with 0.9% NaCl solution and dried at 56° C. for a day.Then each of the samples was hydrolysed in concentrated chloric acid.The samples were then quantitatively investigated for calcium by meansof atom absorption spectroscopy.

[0120] Determining the Amount of Immobilised Heparin

[0121] The amount of immobilised heparin was determined by means ofelementary analysis in accordance with the difference in the content ofsulfur in unmodified (control sample) and modified sample portions(modified sample).

[0122] The method is based on determining the difference in the level ofsulfur concentration in test samples (modified sample) and controlsamples. The sulfur content in heparin is high and low in collagen. Arise in the sulfur content after modification of the biologicalprosthesis accordingly permits calculation of the amount of heparinimmobilised to the biological prosthesis.

Δ[S]heparin=[S]mod. sample−[S]control sample,

[0123] wherein [S] is the sulfur concentration in [μg/g] dry weight.

[0124] In that respect calculation of the heparin content in the sampleis effected in accordance with the following formula:

Y=Δ[S]/S ₁, wherein Y is the heparin content in the biological material(biological prosthesis) in [mg/g], S₁ is the sulfur content in [μg] in 1mg of heparin.

[0125] Basically any quantitative determination method can be used fordetermining the concentration of the sulfur content.

[0126] In the present case the determination procedure was effected asfollows:

[0127] The method of determining the concentration of the sulfur—whichis contained in the range of between 0.2 and 100% in organic samples—isbased on the titration of an aqueous-organic medium after combustion ofthe sample in an oxygen-bearing flask.

[0128] The sample material (at least 20 g) is firstly cut up with a pairof shears until a thin pulpy consistency is attained. Each sample has 10ml of distilled water poured thereover. The sample is frozen at −55° C.and lyophilised with a stepwise increase in temperature to, +60° C.until dry. The dried sample material obtained in that way is finelyground in an agate mortar until a fine powder is produced.

[0129] 5 mg of the sample material is weighed out on an analyticalbalance with a graduation of 0.0001 g. The weighed sample is burnt in aflask which is filled with gaseous oxygen and contains 10 ml of 6% H₂O₂solution. After the burning operation the combustion products are rinsedwith 5 ml of water and cooled. Added to that solution are 0.25 ml of 2 NHCl, 25 ml of acetone and 2 drops of indicator (0.2% aqueouschlorophosphonazo-III solution(bis-(4-chloro-2-phophonobenzolazo)-2,7-chromotropic acid, Fluka ChemieAG, CH-9471 Buchs, Switzerland). Titration is effected with 0.02 NBa(NO₃)₂ solution until there is a transition from a violet-rosecoloration to a light blue coloration. For control purposes a blind testis carried out under analysis conditions including combustion andtitration.

[0130] The sulfur content X is calculated as follows:${X = {\frac{\left( {V - V_{0}} \right) \times K}{A} \times 1000}},$

[0131] wherein:

[0132] V is the volume of 0.02 N Ba(NO₃)₂ solution in [ml], which isused for the titration procedure,

[0133] V₀ is the volume of 0.02 N Ba(NO₃)₂ solution in [ml], which isused for the titration procedure in the blind test,

[0134] K is the conversion factor which reproduces the titer of theBa(NO₃)₂ solution for the sulfur equivalent in [mg/ml], and

[0135] A is the weight of the sample in [mg].

[0136] It will be appreciated that the amount of immobilised heparin canalso be determined in another fashion. For example, it is possible toimplement quantification of immobilised heparin using toluidine bluewhich binds to immobilised heparin.

[0137] Both methods lead to the same result.

RESULT OF EXAMPLE 1/COMPARATIVE EXAMPLES 1

[0138] TABLE 1 Table 1 shows the relative content of free amino acidresidues (related to 1000 amino acid residues) in pig aortic valve velaDEE + Example of the Amino acid Native tissue GA heparin invention THR27.3 ± 0.2 27.6 ± 0.6 24.5 ± 0.3 23.3 ± 0.5 SER 45.3 ± 0.4 46.7 ± 1.141.6 ± 0.4 39.6 ± 0.4 GLU 97.7 ± 0.0 103.0 ± 0.5  89.1 ± 0.7 88.2 ± 1.2OHPRO 110.4 ± 1.2  117.6 ± 1.2  115.6 ± 3.9  110.8 ± 1.8  PRO 25.7 ± 0.528.6 ± 0.4 65.6 ± 0.6 64.8 ± 1.7 GLY 238.2 ± 1.2  252.1 ± 4.1  261.1 ±2.4  272.7 ± 3.2  ALA 124.3 ± 0.9  127.7 ± 0.8  122.3 ± 0.5  132.2 ±3.1  VAL 44.3 ± 0.8 41.9 ± 1.2 42.0 ± 1.7 37.1 ± 1.4 MET 10.7 ± 0.3 10.6± 0.2 — — ILE 19.9 ± 0.3 20.2 ± 0.3 16.0 ± 0.3 16.7 ± 0.5 LEU 42.4 ± 0.342.6 ± 0.6 36.2 ± 0.3 36.1 ± 0.5 TYR 10.4 ± 0.3  9.4 ± 0.2  8.7 ± 0.2 1.9 ± 0.5 PHE 21.9 ± 0.4 20.9 ± 0.4 22.6 ± 0.7 19.7 ± 1.4 HIS 12.9 ±0.2 15.8 ± 0.3 35.6 ± 0.4 39.2 ± 0.6 OHLYS 11.0 ± 0.2  1.4 ± 0.2  2.3 ±0.2 — LYS 34.0 ± 0.3  3.3 ± 0.2  7.4 ± 0.2 — ASP 66.5 ± 0.5 70.0 ± 0.561.2 ± 0.5 60.1 ± 0.9 ARG 57.1 ± 0.8 63.1 ± 1.2 47.8 ± 0.9 56.3 ± 1.3

[0139] It is known that epoxide compounds react with methionine,tyrosine, lysine and hydroxylysine of the biological material, whereasglutaraldehyde reacts only with the last two amino acids. The resultsdemonstrated confirm this.

[0140] In this respect when using the conserving agent according to theinvention the density of transverse cross-linking is greater than whenusing the individual substance—ethylene glycol diglycidylether. That canbe seen in particular from a reduction in the relative content of theamino acids methionine, tyrosine, lysine and hydroxylysine. TABLE 2Table 2 shows the physical-mechanical parameters of pig aortic valvevela conserved with various methods. Conserving agent σ[kg/cm²] ε[%]h[cm] Glutaraldehyde (GA) 59.2 ± 4.6 38.7 ± 1.9 0.059 ± 0.003 DEE +heparin 69.9 ± 5.9 38.7 ± 1.8 0.046 ± 0.002 Example of the inv. 93.5 ±8.0 35.9 ± 1.6 0.041 ± 0.002

[0141] The sample produced according to the example of the invention,with a smaller thickness, has a better tensile strength characteristic(greater breaking stress). TABLE 3 Table 3 shows the amount of calcium(mg/g of dry tissue) in valve vela portions implanted under the skin ofrats at various periods after implantation. Implantation period GA DEE +heparin Example of the inv. Without  2.25 ± 0.10 2.18 ± 0.08  2.23 ±0.11 implantation 30 days 125.6 ± 10.2 2.5 ± 0.07 2.15 ± 0.08 60 days211.7 ± 12.4 2.8 ± 0.10  2.3 ± 0.07 90 days 265.4 ± 21.3 2.5 ± 0.09  2.7± 0.11

[0142] The sample produced in accordance with the example of theinvention has an extremely low degree of calcification. TABLE 4 Table 4shows the amount of immobilised heparin. DEE + heparin Example accordingto the invention 530 ± 20 μg/g dry tissue 2340 ± 120 μg/g dry tissue

[0143] The sample produced in accordance with the example of theinvention has a very high content of immobilised heparin.

EXAMPLE 2 ACCORDING TO THE INVENTION

[0144] Segments of the internal thoracic artery of a pig (25 g of moisttissue) were rinsed with 0.9% by weight NaCl solution and put into 200ml of a conserving solution which is produced from 50 mM HEPES-buffer pH7.4 and contains 8 g of ethylene glycol diglycidylether, 0.5 g ofpolyethylene glycol diglycidylether (n=6) and 1.5 g of pentaerythrolpolyglycidylether (number of glycidylether units per molecule: 4).

[0145] After 48 hours the conserving solution was replaced by anidentical but freshly produced solution. After 12 days the segments wererinsed with sterile 0.9% by weight NaCl solution and incubated in aheparin solution (100 IU/ml) pH 5.0, wherein the pH-value was adjustedby the addition of aqueous acetylsalicylic acid solution (production,see Example 1 according to the invention), for a period of 4 hours at atemperature of 20° C. Production of the heparin solution was effected asdescribed in Example 1.

[0146] Thereafter the segments were rinsed three times with an excess of0.9% by weight NaCl solution and put into a 5% by weight ethylene glycoldiglycidylether solution, where they were stored until further use.

COMPARATIVE EXAMPLES 2

[0147] The comparative samples used were segments of the internalthoracic artery of a pig, which were treated with 0.625% by weightglutaraldehyde in 50 mM phosphate buffer pH 7.4 (GA) and with 5% byweight ethylene glycol diglycidylether and 100 IU/ml heparin in 50 mMphosphate buffer pH-value 7.4 (DEE+heparin) (see RU 2 008 767).Production of the comparative samples was effected in accordance withthe description set forth in relation to comparative Examples 1.

RESULT EXAMPLE 2/COMPARATIVE EXAMPLES 2

[0148] The operation of determining the density of transversecross-linking, the degree of calcification and the amount of immobilisedheparin was effected as described hereinbefore in relation to Example 1.The results contained in Example 1 and comparative Examples 1 areconfirmed by the results obtained in the present case.

[0149] The resistance to thrombosis formation was assessed after theimplantation of vessel segments which were conserved in accordance withthe method according to the invention (example according to theinvention) or for comparison purposes using glutaraldehyde (GA) orethylene glycol diglycidylether and heparin (DEE+heparin), into thecarotid artery of dogs.

[0150] In that case the vessel segments were between about 3 mm and 3.5mm in diameter and between about 5 and 7 cm in length and were implantedin the neck artery (carotid) of 24 non-thoroughbred dogs which weighedbetween 10 and 15 kg.

[0151] The dogs were previously anethetised by the intravenousadministration of sodium thiopental and mechanically ventilated. Acarotid artery segment of between 5 and 7 cm in length was removed fromthe dogs and the bioprosthesis was implanted in place thereof using 6-0polypropylene suture material.

[0152] Eight animals received a ‘GA’ bioprosthesis in the right carotidartery and a ‘DEE+heparin’ prosthesis in the left carotid artery; eightfurther animals received a ‘DEE+heparin’ bioprosthesis in the rightcarotid artery and a bioprosthesis according to the invention in theleft carotid artery; eight further animals received a bioprosthesisaccording to the invention in the right carotid artery and a ‘GA’prosthesis in the left carotid artery. The through-flow in theprostheses was examined prior to closure of the wound by means ofpulsation palpation. The penetration capability of the bioprostheses wasdetermined by means of angiography and ultrasound methods (Dopplerechography, duplex scanning).

[0153] Evaluation of the data obtained was effected by means ofactuarial analysis. Actuarial analysis is a standardised statisticalprocedure which is based on the probability of analysed complications,in the present case thrombosis, in which respect events which havealready occurred are taken into consideration. Evaluation of the resultswas effected with the program STATISTICA for Windows (StatSoft, Inc,1995).

[0154] The actuarial values in respect of penetration capability in [%]of biological prostheses implanted in the neck artery (carotid) of thedogs were determined and are set forth in Table 5. The values specifiedin Table 5 are shown in graph form in FIG. 1. TABLE 5 Table 5 shows theactuarial values of penetration capability of implanted bioprostheses in[%]. Period in DEE + Example months GA heparin of the inv. 0 100% 100%100% 1 75% 97% 98% 2 70% 90% 98% 3 35% 88% 95% 4 15% 86% 90% 5 5% 80%88% 6 0% 76% 86% 7 73% 86% 8 72% 86% 9 70% 80% 10 68% 80% 11 65% 78% 1265% 78%

[0155] TABLE 6 Table 6 shows the relative content of free methionine,tyrosine, lysine and hydroxylysine residues (related to 1000 amino acidresidues) in samples of the internal thoracic artery of a pig. NativeEx. of the Amino acid tissue GA DEE + heparin inv. MET 7.5 ± 0.5 7.3 ±0.4 6.3 ± 0.2 — TYR 10.0 ± 0.6  11.0 ± 0.4  — 3.4 ± 0.5 OHLYS 7.0 ± 0.51.0 ± 0.2 0.9 ± 0.2 — LYS 24.1 ± 1.9  3.5 ± 0.5 — —

[0156] TABLE 7 Table 7 shows the amount of immobilised heparin. DEE +heparin Example according to the invention 190 ± 10 μg/g dry tissue 1005± 90 μg/g dry tissue

[0157] TABLE 8 Table 8 shows the amount of calcium (mg/g of dry tissue)in samples implanted under the skin of rats of the internal thoracicartery of a pig at various periods after implantation. Implantationperiod GA DEE + heparin Ex. of the inv. Without 1.39 ± 0.11 1.5 ± 0.031.42 ± 0.09  implantation 30 days 52.6 ± 4.1  1.35 ± 0.04  1.5 ± 0.10 60days 74.1 ± 9.3  1.8 ± 0.07 1.9 ± 0.07 90 days 92.0 ± 10.4 1.5 ± 0.041.5 ± 0.01

EXAMPLE 3 ACCORDING TO THE INVENTION

[0158] Pig pericardium (30 g of moist tissue) was mechanically cleanedof the surrounding tissue, rinsed with 0.9% by weight NaCl solution andincubated in 200 ml of a conserving solution which is produced from 50mM HEPES-buffer pH 7.4 and contains 4.5 g of ethylene glycoldiglycidylether, 1.5 g of polyethylene glycol diglycidylether (n=4) and4 g of pentaerythrol polyglycidylether (number of the glycidyletherunits per molecule: 4).

[0159] After 48 hours the solution was replaced by an identical abutfreshly produced solution. After 12 days the pericardium was rinsed withsterile 0.9% by weight NaCl solution and incubated in a heparin solution(100 IU/ml) pH 6.0, wherein the pH-value was adjusted by the addition ofaqueous acetylsalicylic acid solution (production, see Example 1according to the invention), for a period of 4 hours at a temperature of20° C.

[0160] Thereafter the pericardium was rinsed three times with an excessof 0.9% by weight NaCl solution and put into a 5% by weight ethyleneglycol diglycidylether solution, where it was stored until further use.

COMPARATIVE EXAMPLES 3

[0161] The comparative samples used were pig pericardium portions whichwere treated with 0.625% by weight glutaraldehyde in 50 mM phosphatebuffer pH 7.4 (GA) or with 5% by weight ethylene glycol diglycidylether(DEE) and 100 IU/ml heparin in 50 mM phosphate buffer pH-value 7.4(DEE+heparin) (see RU 2 008 767). Production of the comparative sampleswas effected in accordance with the description set forth in relation tocomparative Examples 1.

[0162] The operation of determining the density of transversecross-linking, the properties in respect of elasticity and deformationand the amount of immobilised heparin was effected as described inExample 1. The results obtained in Example 1 and comparative Examples 1are confirmed by the results obtained in the present case.

RESULT OF EXAMPLE 3 ACCORDING TO THE INVENTION/COMDPARATIVE EXAMPLES 3

[0163] TABLE 9 Table 9 shows the relative content of free methionine,tyrosine, lysine and hydroxylysine residues (related to 1000 amino acidresidues) in pig pericardia. Ex. of the Amino acid native tissue GADEE + heparin inv. MET 8.8 ± 0.5 8.7 ± 0.5 4.4 ± 0.3 — TYR 8.2 ± 0.2 8.0± 0.4 — 5.0 ± 0.3 OHLYS 9.9 ± 0.4 1.1 ± 0.1 0.7 ± 0.2 1.5 ± 0.1 LYS 32.0± 1.9  2.8 ± 0.1 — 1.4 ± 0.3 ARG 55.6 ± 0.8  58.6 ± 0.5  50.9 ± 0.8 12.7 ± 0.3 

[0164] TABLE 10 Table 10 shows the amount of immobilised heparin. DEE +heparin Example according to the invention 760 ± 10 μg/g dry tissue 2640± 85 μg/g dry tissue

[0165] TABLE 11 Table 11 shows the physical-mechanical parameters of pigpericardium portions conserved with various methods. Conserving agent σ[kg/cm²] ε [%] h [cm] Glutaradehyde 119.8 ± 6.2 48.5 ± 5.8 0.046 ± 0.003DEE + heparin 123.3 ± 11.4 58.4 ± 3.5 0.044 ± 0.004 Ex. of the inv.125.5 ± 12.3 57.0 ± 3.6 0.045 ± 0.005

1. A method of conserving biological prostheses, characterised in thatthe method includes the following steps: (a) treating biologicalprostheses with a solution which contains a mixture of epoxide compoundswhich are at least in part of different lengths; (b) treating thebiological prosthesis treated in accordance with step (a) with anantithrombotic-bearing solution; and (c) possibly storing the prosthesistreated in accordance with step (b) in a sterilising solution.
 2. Amethod as set forth in claim 1 characterised in that step (a) involvesusing a solution which contains a mixture of at least three differentepoxide compounds.
 3. A method as set forth in claim 1 or claim 2characterised in that the mixture of epoxide compounds includes at leastone non-polymer epoxide compound with two epoxide groups.
 4. A method asset forth in one of the preceding claims characterised in that themixture of epoxide compounds includes at least one polymer epoxidecompound with between two and three epoxide groups and/or an epoxidecompound with between two and three epoxide groups, wherein arrangedbetween at least two epoxide groups is a straight-chain or branchedhydrocarbon chain with at least four carbon atoms.
 5. A method as setforth in one of the preceding claims characterised in that the mixtureof epoxide compounds includes at least one epoxide compound with atleast three epoxide groups.
 6. A method as set forth in one of thepreceding claims characterised in that the epoxide group is a componentof a glycidol residue.
 7. A method as set forth in claim 3 characterisedin that the non-polymer epoxide compounds are selected from the groupwhich consists of alkylene glycol diglycidylether, in particularethylene glycol diglycidylether, alkane diol diglycidylether, inparticular butane-1,4-diol diglycidylether, polyalcohol diglycidylether,in particular glycerine diglycidylether, and mixtures thereof.
 8. Amethod as set forth in claim 4 characterised in that the polymer epoxidecompounds are selected from the group which consists of polyalkyleneglycol diglycidylether, in particular polyethylene glycoldiglycidylether, polytetramethylene glycol glycidylether, polypropyleneglycol diglycidylether and mixtures thereof.
 9. A method as set forth inclaim 4 characterised in that the epoxide compound used is alkane dioldiglycidylether, in particular hexane-1,6-diol diglycidylether, and/ordicarboxylic acid diglycidylester.
 10. A method as set forth in claim 6characterised in that the epoxide compounds with at least three epoxidegroups are selected from the group which consists of polyalcoholpolyglycidylether, in particular sorbitol polyglycidylether, glycerinepolyglycidylether, pentaerythrol polyglycidylether, polysaccharidepolyglycidylether and mixtures thereof.
 11. A method as set forth in oneof the preceding claims characterised in that the antithrombotic isselected from the group which consists of heparin, low-molecularheparin, heparinoids, hirudin and mixtures thereof.
 12. A method as setforth in one of the preceding claims characterised in that theantithrombotic is a mixture of heparin and acetylsalicylic acid.
 13. Amethod as set forth in claim 12 characterised in that after thetreatment in accordance with step (a) in step (b) the biologicalprosthesis is treated with a solution of heparin and acetylsalicylicacid without using additional reagents.
 14. A method as set forth inclaim 12 or claim 13 characterised in that after the treatment withheparin and acetylsalicylic acid in accordance with step (b) thebiological prosthesis is rinsed in distilled water or an isotonicsolution, in particular a 0.9% by weight NaCl solution, and thensterilised by treatment with a solution of any epoxide compound.
 15. Amethod as set forth in claim 14 characterised in that the solution usedfor sterilisation is of a concentration at the epoxide compound of atleast 2% by weight.
 16. A method as set forth in one of the precedingclaims characterised in that the biological prosthesis is produced fromthe heart valves of mammals.
 17. A method as set forth in one of thepreceding claims characterised in that the biological prosthesis isproduced from vein valves or valve-containing vein segments of mammals.18. A method as set forth in one of the preceding claims characterisedin that the biological prosthesis is produced from artery segments ofmammals.
 19. A method as set forth in one of the preceding claimscharacterised in that the biological prosthesis is produced frommembranous tissues, in particular pericardium or hard meninges, ofmammals.
 20. A conserved biological prosthesis characterised in that thebiological prosthesis is produced in accordance with a method as setforth in one of claims 1 through
 19. 21. A conserved biologicalprosthesis as set forth in claim 20 characterised in that the biologicalprosthesis is a heart valve, an artery segment, a vein segment,membranous tissue, in particular pericardium or hard meninges of amammal.
 22. A conserving solution for biological prosthesescharacterised in that the conserving solution contains a mixture ofepoxide compounds which are at least in part of different lengths.
 23. Aconserving solution as set forth in claim 22 characterised in that theconserving solution contains a mixture of at least three differentepoxide compounds.
 24. A conserving solution as set forth in claim 22 orclaim 23 characterised in that the conserving solution includes at leastone non-polymer epoxide compound with two epoxide groups.
 25. Aconserving solution as set forth in one of claims 22 through 24characterised in that the conserving solution contains at least onepolymer epoxide compound with between two and three epoxide groupsand/or at least one epoxide compound with between two and three epoxidegroups, wherein arranged between at least two epoxide groups is astraight-chain or branched hydrocarbon chain with at least four carbonatoms.
 26. A conserving solution as set forth in one of claims 22through 25 characterised in that the conserving solution contains atleast one epoxide compound with at least three epoxide groups.
 27. Aconserving solution as set forth in one of claims 22 through 26characterised in that the conserving solution, in relation to the totalamount of epoxide compounds, contains 40-80% by weight, preferably50-70% by weight, of at least one non-polymer epoxide compound with twoepoxide groups; 5-20% by weight, preferably 10-15% by weight, of atleast one polymer epoxide compound with between two and three epoxidegroups and/or at least one epoxide compound with between two and threeepoxide groups, wherein arranged between at least two epoxide groups isa straight-chain or branched hydrocarbon chain with at least four carbonatoms; and 15-45% by weight, preferably 20-35% by weight, of at leastone epoxide compound with at least three epoxide groups; wherein thetotal amount is 100% by weight.